Many others are currently interested in developing assay systems which will permit the direct detection of viruses in biological specimens. Most approaches seem to be modeled on the double antibody sandwich similar to that found to be effective for rotavirus detection. For that purpose antirotavirus antibody is adsorbed on a bead of size convenient for handling. The antirotavirus antibody bead is then exposed to a stool specimen suspected of containing rotavirus. Following exhaustive washing of the bead any rotavirus present is "sandwiched" with a second antirotavirus antibody to which enzyme has been conjugated. An enzyme assay is then performed to detect the presence of viral particles.
Nearly all of the reports described assay systems which exploit the double-antibody sandwich (DAS) approach with conjugation of .sup.125 I or enzyme (alkaline phosphatase, or horseradish peroxidase) to the second antibody, or indirect analysis, which involves the use of a third antibody to which an enzyme or radiolabel has been conjugated. In a limited number of instances competitive inhibition with labelled or quantified antigen has been suggested along with DAS.
The inventors are also aware of one report of direct adsorption of viral antigen to a substrate used to detect intact picornavirus. This approach can be sucessful with picornaviruses if there is no competing protein antigen in the system.
A severe limitation in detecting virus particles through assay for surface antigens is the widespread existence of proteases as in biological specimens. If samples are not immediately frozen and maintained in a frozen state, surface antigens may be degraded and rendered non-reactive with the antibodies intended to detect them.
Two reports of influenza virus detection systems appear in the literature. The first method (Berg et al., 1980) involves a minimum of 24 hours to carry out and uses fluorescent or radioactive substrates to indicate the presence of influenza virus. The procedure is quite complicated and requires special equipment to detect the fluorescence or radioactivity of the indicator. A second approach (Yolken, et al., 1980) employs "capture" antibody on a Microtiter plate to adsorb neuraminidase in intact viral particles. A neuraminidase assay with a fluorescent substrate (methyl umbelliferyl neuraminic acid) indicates the presence of virus. However, the neuraminidase antigen (particularly Nl) varies greatly in its stability as an enzyme among different subtypes. Inactivation of this enzyme prevents analysis. Further limitations associated with this assay are those arising from its dependence on fluorometry (which precludes the use of visual analysis), and the occurrence of non-specific reactions which yield spurious results.